Purification of rat liver phosphorylase kinase.
A rapid method for the purification of rat liver phosphorylase kinase 30,000-fold over homogenate values is described. The method allows the isolation of a near homogeneous preparation of phosphorylase kinase initially associated with the glycogen pellet to be accomplished within 24 h. The enzyme has Mr (apparent) = 1.3 million by gel filtration and is composed of subunits similar in size to those of skeletal muscle phosphorylase kinase. The enzyme is phosphorylated by the cAMP-dependent protein kinase: phosphate is incorporated into two of the subunits (Mr = 140,000 and Mr = 116,000) and is closely paralleled by activation of the enzyme. The enzyme is partially inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and is stimulated by 10(-8)-10(-6) M Ca2+. The pH optimum of the nonactivated enzyme is 7.0. Activation by cAMP-dependent protein kinase does not appear to alter the Ca2+ sensitivity of the enzyme. However, it results in a large increase in activity at pH 7 through 8, but not at pH below 6.5. Purified rat liver phosphorylase kinase thus shows many similarities to purified skeletal muscle phosphorylase kinase, but differs in respect to its incomplete inhibition by ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid and to the effects of phosphorylation by cAMP-dependent protein kinase on its pH activity profile and Ca2+ sensitivity.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010764:Phosphorylase Kinase / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats
D046911:Macromolecular Substances; D004533:Egtazic Acid; D010764:Phosphorylase Kinase; D002118:Calcium